1. 1 The physical, chemical, and biological properties of small molecules can be profoundly influenced by crystal form. -Ostwald, W. Zeitschrift fur Physikalsche Chemie 1897, 22, 289-330. Wilhelm Ostwald predicted adiabatic transitioning through metastable crystal polymorphs en route to the thermodynamic energy minimized structure. Garth J. Simpson Purdue University

2. 2 ? G + - Gibbs free energy (ΔG ) ΔG s ΔG v radius (r) ΔG * 3D r * 3D Metastable forms can exhibit lower free energy barriers for crystal nucleation For fast crystallization, the metastable form may be kinetically trapped. -Ostwald, W. Zeitschrift fur Physikalsche Chemie 1897, 22, 289-330. -Hall, V. J.; Simpson, G. J., J. Am. Chem. Soc. 2010, 132, 13598-13599.

3. SHG: Second harmonic generation Reflection & birefringence Linear OpticsNonlinear Optics (NLO)

4. 4 SHG-activity of racemic proline samples prepared by physically mixing homochiral crystals vs. slow crystallization from a racemic solution. Physical MixtureSlow Dry Laser transmittance SHG 100 µm

5. 5 A.U. Chowdhury, C.M. Dettmar, S. Z. Sullivan, S.Zhang, K. T. Jacobs, D. J. Kissick, T. Maltais, G. J. Simpson accepted for publication in JACS. Phase boundary calculated using just the two endpoint solubilities (homochiral and racemic) based on extension of a model developed by Blackmond and coworkers (Angew. Chem. Int. Ed. 2006, 45, 7985–7989.)

6. 6  Metastable proline crystals form upon rapid drying Physical MixtureSlow DryInkjet printed Laser transmittance SHG 100 µm

7.  ~1 pL of solution / droplet  A few pg of residual solid material  ~10  m printed spot size  On the order of 10 9 – 10 11 molecules.  A whole host of possible crystalline and amorphous forms. 7

8.  XRD is the gold standard for structure determination.  Can we assess identify crystal positions by XRD? 8 Alas. Conventional X- ray diffractometers do not have sufficient S/N to enable microcrystal detection/analysis of pg quantities of material present in the inkjet printed dots.

9.  Argonne National Lab is just south of Chicago. 9 Argonne National Laboratory, Argonne IL.

10. 10  A class 4 laser system in the X-ray experimental hutch at a national laboratory.  Small footprint (1 ft 2 ).  <1 um precision in positioning.  Fast acquisition (few s) with low limits of detection.  Software integration.  Long times between required service.  Automated transition from SHG to TPE-UVF.

11.  Droplets were inkjet printed onto low-background mounts and analyzed by SHG+XRD.  1s integration times for XRD. 11 100 µm Diffraction pattern from a single sub-fg printed droplet.

12.  Azimuthal (vertical) autocorrelation selects spots and suppresses diffuse scattering. 12 22  Remapping to polar coordinates. Intensity (a.u.) 2  (deg.) Powder pattern equivalent from a 5µm×5µm×1µm volume

13. 13  Several diffraction peaks agree between the L-proline droplets and the racemic SHG-active droplets 2θ2θ Racemic SHG Silent Racemic SHG Active L proline A.U. Chowdhury, C.M. Dettmar, S. Z. Sullivan, S.Zhang, K. T. Jacobs, D. J. Kissick, T. Maltais, G. J. Simpson accepted for publication in JACS.

14.  In the SHG-active racemic droplets, 64% of the diffracted intensity coincides with peak locations observed for the L-proline droplets.  In the SHG-inactive racemic droplets, 1% of the diffracted intensity coincides with peak locations observed for the L-proline droplets.  Polarization-dependent SHG measurements support the formation of homochiral crystals from inkjet printing of racemic solutions.  Rapid crystallization appears to support the formation of metastable homohiral crystal forms.  Metastable crystallization under kinetic control could provide a route for homochiral amplification through crystallization.  Combined SHG+XRD can dramatically lower detection limits in powder XRD. 14

15. 15 Collaborators Alex Wei Thora Maltais Argonne / APS Kevin Battaile Michael Becker Robert Fischetti Lisa Keefe Steve Ginell Anne Mulichak Funding Integrated system: Jeremy Madden Chris Dettmar Justin Newman SHGM of GPCR Xtals in LMPs: David Kissick NIH R01GM103401 NIH R01GM103910 Chris Dettmar Scott Toth Justin Newman Azhad Chowdhury

16. NIH R01GM103401 NIH R01GM103910 Collaborators: Argonne / APS Kevin Battaile Michael Becker Robert Fischetti Lisa Keefe Steve Ginell Anne Mulichak Purdue Alex Wei Thora Maltais JAFCI R. Michael Everly Hartmut Hedderich Robert Oglesbee